Ligation Enhancement

ABSTRACT

Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double-stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.

CROSS REFERENCE

This application claims priority from U.S. Provisional Application No.61/483,348 filed May 6, 2011, herein incorporated by reference.

BACKGROUND

Double-stranded nucleic acids containing staggered ends, blunt ends orterminal overhangs of one or two bases can be joined by means ofintermolecular or intramolecular ligation reactions. However, ligationof polynucleotides with blunt ends or with one or two base overhangs hasbeen found to be very inefficient. It has been reported thatpolyethylene glycol (PEG) 6000 can enhance ligation of blunt-endeddouble-stranded polynucleotides but this reagent interferes withsubsequent electroporation of competent cells, a common step in cloningand library preparation.

Using restriction endonuclease-cleaved DNA to create staggered ends,Hayashi et al. (Nucleic Acids Research 14(19):7617-7630 (1986)) ligateddouble-stranded DNA with a four-base overhang using T4 DNA ligase and6%-10% (w/v) PEG 6000 to enhance intramolecular ligation and 10% PEG6000 containing divalent cations or polyamines to enhance intermolecularligation. Pheiffer et al. (Nucleic Acids Research 11(22):7853-71 (1983))reported that polymers such as PEG, bovine serum albumen or glycogencould stimulate blunt-end ligation generated by restriction endonucleasecleavage using T4 DNA ligase. In this reference, the authors reportedthat smaller molecules PEG 200 or PEG 400 had little effect on ligation.Xiao et al. (Molecular Biotechnology 35: 129-133 (2007)) investigatedthe effects of the organic compounds dimethyl sulfoxide (DMSO),Tween®-20 (Uniqema America LLC, Wilmington, Del.), glycerol, formamideand PEG 6000 on the efficiency and specificity of ligase-detectionreactions. They reported that all these compounds except PEG 6000inhibited the efficiency of ligation although at low concentrations(0-1%) there was a boost in efficiency using Tween-20 which thendecreased dramatically. DMSO, glycerol and formamide inhibited theefficiency of the ligation reaction at nick junctions in double-strandedDNA.

Blunt-ended or staggered-ended double-stranded polynucleotides arecommonly the product of restriction endonuclease-digestion orfragmentation and polishing. Double-stranded polynucleotides with asingle-base overhang are commonly products of PCR amplification usingnon-proofreading polymerases or the product of adding a dA to ablunt-ended fragment. Alternatively, polishing of the single-baseoverhang can result in a blunt end. Intermolecular ligation of arestriction endonuclease-cleaved polynucleotide or PCR-amplifiedpolynucleotide to a vector is commonly required prior to a cloning stepinvolving transformation of competent cells.

Intermolecular ligation of single-stranded polynucleotides is undertakenin methods which, for example, involve the ligation of barcode sequencesto single-stranded polynucleotides such as cDNA or RNA for cDNAlibraries (Edwards et al. Nucleic Acids Research 19(19):5227-32 (1991))or microRNA cloning (Aravin A, et al. FEBS Lett. 579(26):5830-40 (2005).Epub 2005)) or for sequencing or multi-array techniques. It would bedesirable to improve the efficiency of intermolecular and intramolecularligations of single-stranded and double-stranded polynucleotides withoutinhibiting downstream reactions.

SUMMARY

In general in a first aspect, a composition includes a ligase and one ormore small molecule ligation enhancers having a molecular weight of lessthan 1000 daltons.

Various embodiments include one or more of the following features:

-   -   a ligase reaction buffer contains a small molecule enhancer        having a concentration of 1%-50% v/v that is capable of        enhancing, by at least 25%, intramolecular or intermolecular        ligation of a polynucleotide fragment or fragments than would        otherwise be obtained in the absence of the enhancer;    -   the one or more small molecule ligation enhancers are selected        from an optionally substituted straight or branched chain diol        or polyol containing 2 to 20 carbons, alcohols, zwitterionic        compounds and polar aprotic molecules;    -   the optionally substituted straight or branched diol or polyol        is selected from the group consisting of 1,2-ethylene glycol,        1,2-propanediol (1,2-PrD), 1,3-propanediol (1,3-PrD), glycerol,        pentaerythritol, sorbitol, diethylene glycol, dipropyleneglycol,        neopentyl glycol, 2-methyl-1,3-propanediol,        2,2-dimethyl-1,3-propanediol, 1,3-butanediol, and 1,4-butanediol        1,5-pentanediol, 1,6-hexanediol, 1,9-nonanediol,        1,12-dodecanediol, 2,2′-dimethylpropylene glycol,        1,3-butylethylpropanediol, methylpropanediol,        methylpentanediols, propylene glycol methyl ether, dipropylene        glycol methyl ether, tripropylene glycol methyl ether, propylene        glycol isobutyl ether, ethylene glycol methyl ether, ethylene        glycol ethyl ether, ethylene glycol butyl ether, diethylene        glycol phenyl ether, and propylene glycol phenol ether;    -   the alcohol is an optionally substituted straight or branched        aliphatic primary, secondary, tertiary alcohol chain alkylene        group or polyvalent branched chain alkyl group, containing from        2 to 20 carbons;    -   the zwitterionic compound is optionally substituted, straight or        branched, containing from 2 to 50 carbons and further includes        an ammonium, phosphonium, or sulphonium cationic group, a        carboxylate, phosphate or sulfate anionic group;    -   the polar aprotic molecule is selected from the group consisting        of N-alkylcaprolactams, dimethylcaprylic/capric amides,        N-alkylpyrrolidones, diphenyl sulfone, DMSO, N,N′-dimethyl        imidazolidin-2-one (DMI), acetonitrile, acetone, diglyme,        tetraglyme, tetrahydrofuran (THF), dimethylacetamide, and        dimethylformamide (DMF);    -   the one or more small molecule enhancers comprise a propylene        glycol and an ethylene glycol ether;    -   the one or more small molecule enhancers are selected from the        group consisting of: 1,2-ethylene glycol, 1,2-PrD, 1,3-PrD,        isopropyl alcohol, 1,4-butanediol, 1,5-pentanediol,        1,6-hexanediol;    -   the small molecule enhancer is selected from the group        consisting of: a quaternary ammonium or phosphonium cation and a        carboxylate anion;    -   the small molecule enhancer is selected from the group        consisting of: 1,2-PrD, 1,3-PrD, ethylene glycol, ethanol,        isopropanol, and betaine;    -   the small molecule enhancer is DMSO;    -   the small molecule enhancer is 1,2-PrD;    -   the composition further includes PEG;    -   the amount of the small molecule enhancer is in the range of        2%-20% v/v;    -   at least two nucleic acid fragments wherein the nucleic acid        fragments are double-stranded DNA having blunt ends for ligation        to each other and the ligase is a DNA ligase;    -   at least two nucleic acid fragments where the nucleic acid        fragments are double-stranded DNA having single-base overhangs        for ligation to each other; and the ligase is a DNA ligase;        and/or    -   the DNA ligase is selected from the group consisting of T3 DNA        ligase, T4 DNA ligase, T7 DNA ligase, Taq Ligase, Ampligase®, E.        coli ligase and a Sso7-ligase fusion.

In general in a second aspect, a reaction mixture is provided thatincludes a ligase, one or more small molecule ligation enhancers, and aligation buffer in the absence of PEG.

In general in a third aspect, a method is provided of enhancing ligationbetween nucleic acid fragments, the method including: mixing a smallmolecule ligation enhancer with a molecular weight of less than 1000daltons with a plurality of nucleic acid fragments and a ligase in aligation buffer; and permitting ligation wherein the efficiency ofligation is enhanced by at least 25% compared to the efficiency of aligation in the absence of the small molecule enhancer.

Various embodiments include one or more of the following features:

-   -   ligation is intramolecular;    -   ligation is intermolecular;    -   the plurality of nucleic acid fragments are double-stranded DNA        with blunt ends, single-base overhangs or staggered ends; and/or    -   the efficiency of ligation is enhanced by at least 4-fold as        determined by electroporation of host cell.

In general in a fourth aspect, a composition is provided that includes0.01-200 units/μl restriction endonuclease, 0.01-2000 units/μl ligase,0.01-200 units/μl polynucleotide kinase (PNK), in a buffer containingone or more small molecule ligation enhancers with a molecular weight ofless than 1000 daltons and optionally PEG 5000-10,000.

Various embodiments include one or more of the following features:

-   -   the ligase is T4 ligase;    -   the one or more small molecule ligation enhancers comprise        1,2-PrD; and/or    -   the one or more small molecule ligation enhancers are 1,2-PrD        and glycerol.

In general in a fifth aspect, a method is provided for creating apolynucleotide library that includes: adding 0.01-200 units/μlrestriction endonuclease, 0.01-2000 units/μl ligase, and 0.01-200units/μl PNK in a buffer containing one or more small molecule ligationenhancers and optionally PEG 5000-10,000 to a polynucleotideamplification product; and allowing intramolecular ligation to occur fortransforming competent host cells.

An embodiment includes transforming competent cells usingelectroporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the results of recircularization through ligation ofdouble-stranded linearized plasmid DNA in the presence of increasingamounts of 1,2-PrD. The ligated DNA is subsequently transformed intocompetent E. coli cells, followed by plating on agar ampicillinselection plates. The extent of successful transformation is illustratedby the numbers of colonies on the plate. The greater the 1,2-PrDconcentration, the greater the number of transformants.

FIG. 1A shows the product of ligation in the absence of 1,2-PrD.

FIG. 1B shows the product of ligation in the presence of 4% 1,2-PrD.

FIG. 1C shows the product of ligation in the presence of 6% 1,2-PrD.

FIG. 2 shows the results of a ligase activity titer assay usingblunt-ended DNA fragments which were the HaeIII restriction endonucleasecleavage products of PhiX-174 genomic DNA. The ligase activity in theabsence and presence of 6% 1,2-PrD was determined in a two-fold ligasedilution series. The symbol “*” denotes equivalent levels of ligation,showing that ligation efficiency was enhanced 4-fold. The first and lastlanes contain DNA markers.

FIGS. 3A-C show how cleavage of pUC19 plasmid with the specifiedrestriction enzyme (AhdI, BciVI and BccI) generates one, two and threefragments which when ligated properly form a circularized plasmid.Recognition sequences for AhdI (SEQ ID NO:1—top and bottom strands),BciVI (SEQ ID NOS: 2 and 3—top and bottom strands) and BccI (SEQ IDNO:4—bottom strand) are shown.

FIG. 3A shows the expected product of ligation of a linearized plasmidwith single-base 3′ overhangs into a circularized vector.

FIG. 3B shows the circularized product of ligation of two fragments witha single-base overhang obtained by cleaving a vector. This is a modelsystem for “insert vector” cloning where the two fragments, each withsingle-base 3′ overhang, are ligated into a circle.

FIG. 3C shows the circularized product of ligation of three fragmentswith single-base 5′ overhangs. Only ligated constructs with all threepieces present and in the correct orientation lead to a viabletransformant and a subsequent colony on an agar ampicillin selectionplate. Assembly of just two fragments does not reconstitute the βlactamase ampicillin resistance gene, and thus does not createampicillin-resistant colonies.

FIGS. 4A-C show agarose gel electrophoresis analysis of ligationproducts achieved using +/−12% 1,2-PrD for one, two and three pUC19fragment sets as described in FIGS. 3A-C. The marker lane indicated by Mcorresponds to 1 μg of a 2-log DNA ladder, New England Biolabs, Inc.(NEB), Ipswich, Mass., #3200. (1) is a 2686-bp linear AhdI-pUC19fragment; (2) are 1527-bp and 1159-bp BciVI fragments; (3) are 2 lengths(2275-bp and 287-bp) of 3 BccI fragments where the third 124-1) byfragment is not visible on the gel; (4) is a linearized intermediateligation product formed by 2275-bp and 287-bp BccI fragments; (5) is thedesired ligated/circularized plasmid which is a highly transformabledesired product; and (6) are different kinds of linear and circularundesired concatameric ligation products which are not highlytransformable.

FIG. 4A refers to Ahdl-pUC19 (see FIG. 3A).

FIG. 4B refers to BciVI-pUC19 (see FIG. 3B).

FIG. 4C refers to BccI-pUC19 (see FIG. 3C).

FIG. 5 shows a histogram comparing DNA ligation (and subsequenttransformation of E. coli) using regular ligation buffer with varyingamounts of 1,2-PrD, without 1,2-PrD and with Quick Ligase™ buffercontaining PEG (NEB, Ipswich, Mass.). The addition of 1,2-PrD to theligation buffer enhanced DNA ligation at least as much as PEG containingQuick Ligase. The Y-axis is transformants per 10 pg DNA (transformationsw/chemically competent cells.) The X-axis is regular ligase buffer (RLB)with the percentage of added 1,2-PrD given in parentheses. The quickligase buffer (QLB) has PEG in it to enhance ligation.

FIG. 6 shows enhanced ligation efficiency of T4 DNA ligase by a smallmolecule enhancer for Next Generation Sequencing (NGS) librarypreparation. Libraries were prepared using either T4 DNA ligase in aligase buffer containing PEG (Quick Ligase buffer, NEB, Ipswich, Mass.)or T4 DNA ligase in a ligase buffer containing a small molecule enhancer(ligation buffer, NEB, Ipswich, Mass.) using 5 ng E. coli genomic DNAfragmented to 200 bp. “No template” control contained all the reagentsexcept for DNA, and went through the whole workflow. Library yields wereincreased at least about 5-fold in the presence of the small ligaseenhancer.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In embodiments of the invention, small molecules having a molecularweight of less than 1000 daltons, 900 daltons, 800 daltons, 700 daltons,600 daltons, 500 daltons or 400 daltons have been identified that serveas ligation enhancers. Examples of small molecule enhancers includestraight or branched diol or polyol containing 2 to 20 carbons. Examplesinclude optionally substituted straight or branched alkylene glycols,pentaerythritol, sorbitol, diethylene glycol, dipropylene glycol,neopentyl glycol, such as propylene glycol and ethylene glycol ethers,such as 1,2-ethylene glycol, 1,2-PrD, 1,3-PrD, 1,4-butanediol,1,5-pentanediol, 1,6-hexanediol, 2-methyl-1,3-propanediol,2,2′-dimethylpropylene glycol, 1,3-butylethylpropanediol, methylpropanediol, methyl pentanediols, propylene glycol methyl ether,propylene glycol ethyl ether, propylene glycol butyl ether, diethyleneglycol phenyl ether, propylene glycol phenol ether, propylene glycolmethyl ether, tri-propylene glycol methyl ether, propylene glycolisobutyl ether, ethylene glycol methyl ether, or mixtures thereof.

Additional examples of small molecule ligation enhancers includealcohols which may be substituted straight or branched and includeprimary, secondary, tertiary alcohol chain alkylene group or polyvalentbranched chain alkyl group containing 2 to 20 carbons such as methanol,ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, i- orneo-pentanol, n-hexanol, n-heptanol, n-octanol, 2-ethylhexanol,n-nonanol and mixtures thereof.

Additional examples of small molecule enhancers include zwitterioniccompounds which are optionally substituted straight or branched andcontain 2 to 50 carbons and comprise an ammonium, phosphonium orsulfonium cationic group and carboxylate, phosphate, or a sulfate groupsuch as sulpho- or carboxy-betaines, amino acids, aminosulfonic acids,and alkaloids.

Additional examples include a polar aprotic molecule selected from thegroup consisting of N-alkylcaprolactams, dimethylcaprylic/capric amides,N-alkylpyrrolidons, diphenyl sulfone, DMSO, DMI, acetonitrile, acetone,diglyme, tetraglyme, THF, dimethylacetamide, and DMF.

One or more of the above-described small molecule enhancers can be addedto a preparation of polynucleotides and one or a plurality of ligases toenhance ligation.

The small molecule enhancers are useful for facilitating ligation ofdouble-stranded nucleic acids that have single nucleotide extensions orhave blunt ends. The enhancers may also be used to enhance any type ofligation including those involving double-stranded nucleic acidscontaining 2-6 bases or even longer extensions or single-strandednucleic acid ligation. The term “nucleic acid” as used herein refers todouble-stranded DNA, double-stranded RNA and double-stranded DNA/RNAmolecules in which one strand is DNA and the other is RNA or part of themolecule is double-stranded DNA and part is double-stranded RNA, orsingle-stranded DNA or RNA.

The term “ligase” as used herein refers to an enzyme that is commonlyused to join polynucleotides together or to join the ends of a singlepolynucleotide. Ligases include ATP-dependent double-strandpolynucleotide ligases, NAD⁺-dependent double-strand DNA or RNA ligasesand single-strand polynucleotide ligases. Present embodiments describeenhancement of ligation where the ligase is selected from any of theligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2(NAD⁺-dependent ligases), EC 6.5.1.3 (RNA ligases) (see ExPASyBioinformatics Resource Portal having a URL of enzyme.expasy.org whichis a repository of information concerning nomenclature of enzymes basedon the recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUBMB)describing each type of characterized enzyme for which an EC (EnzymeCommission) number has been provided. Specific examples of ligasesinclude bacterial ligases such as E. coli DNA ligase and Taq DNA ligase,Ampligase® thermostable DNA ligase (Epicentre®Technologies Corp., partof Illumina®, Madison, Wis.) and phage ligases such as T3 DNA ligase, T4DNA ligase and T7 DNA ligase and mutants thereof. Examples of mutantsinclude fusion ligases containing a DNA-binding domain and a ligase.Examples of fusion ligases include: Sso7-T3 DNA ligase, Sso7-T4 DNAligase, Sso7-T7 DNA ligase, Sso7-Taq DNA ligase, Sso7-E. coli DNA ligaseand Sso7-Ampligase DNA ligase. Other mutants may contain a mutation inthe catalytic domain of the ligase that permits ligation of adenylatedDNA only, such as a mutation of the lysine therein. One example is amutation of lysine at position 159 to any other amino acid in T4 DNAligase (Accession number of NP_(—)049813 at NCBI). Other examplesinclude T4 RNA ligase 1 and T4 RNA ligase 2 and mutants thereof, forexample, Sso7 fusion proteins, T4 truncated and mutated (K227Q) RNAligase.

Advantages of any of the small molecule enhancers added to one or moreof the ligases described herein for joining two or more polynucleotidescan be ascertained using the assays described in the examples or any ofthe ligase assays known in the art, for example, ligase assays describedin the New England Biolabs (Ipswich, Mass.) catalogue 2011/12 on pages172-173 and 108-110. Such assays include: inserting a DNA containing amarker (such as antibiotic resistance) into a vector and cloning theproduct so as to detect the presence of the marker; and assembling 2 or3 or more nucleic acid fragments and introducing the ligation productinto a host cell by transformation to determine expression of aphenotype encoded by the ligated polynucleotide.

In some circumstances, a person of ordinary skill in the art might use aligation buffer additionally containing PEG to facilitate difficultligations. Unfortunately, PEG interferes with electroporation and has tobe removed prior to this step. FIG. 5 shows how difficult ligations maybe achieved in the presence of a small molecule enhancer in the absenceof PEG. This is beneficial not only because the efficiency of ligationis enhanced but also because it avoids the need to purify the DNA awayfrom the PEG prior to electroporation.

In embodiments of the invention, for any ligation reaction, the enhancermay be added to the ligation buffer at a concentration in the range of1%-50% v/v, 2%-50% v/v, 3%-50% v/v, 4%-50% v/v, 5%-50% v/v, 1%-45% v/v,1%-40% v/v, 1%-35% v/v, 1%-30% v/v, 1%-25% v/v, 1%-25% v/v, 1%-15% v/vwith 0.01-2000 units/μl ligase, for example, 1-1000 units/μl ligase or4-200 units/μl ligase. When PEG is present in the ligase buffer, atwo-fold reduction in the small molecule enhancer concentration overthat used in the absence of PEG can be used to further enhance ligationover that seen with PEG alone; for example, where a small moleculeenhancer might be used in a range of 10%-18% v/v in the absence of PEG,5%-9% v/v small molecule enhancer can be used with PEG. In oneembodiment, blunt-end double-stranded nucleic acid ligation utilizes asmall molecule enhancer at about 4% v/v in the absence of PEG. Otherenzymes may be included in the ligase reaction mixture such as arestriction endonuclease such as DpnI, or a PNK such as T4 PNK whereeither enzyme may be added at a concentration in the range of 0.01-200units/μl, for example 0.05-50 units/μl. In one example of a ligationreaction, a mixture of nucleic acid fragments that includes a vector andan insert in the form of separate nucleic acids can give rise to adesired ligation product in the presence of small molecule enhancers. Inparticular, the concentration of the enhancer is selected to favorcircularization over concatamerization both for a single species ofmolecule and for mixtures of nucleic acids such as vector and insertcombinations. It is possible to enhance the desired ligation reaction byat least 25% where for example the overall range of enhancement is atleast 1.25-fold to 75-fold for example, at least 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more for moleculeswith single-base overhangs and by at least 25% for example at least1.25-fold, 2-fold, 3-fold, 4-fold for blunt-ended molecules with orwithout PEG.

Table 1 lists small molecule enhancers of ligation. All were testedside-by-side using two ligation test systems which were: (a) cloning ofa 500 bp amplicon made by Taq DNA polymerase into a T-tailed vector; and(b) recircularization of an AhdI-linearized plasmid. Both ligation testsystems used DNA with single-base overhangs.

TABLE 1 Small Molecule Cloning Recircularization Enhancer EnhancementEnhancement No addition 1-fold 1-fold 1,2-PrD (12%) 14-fold 11-fold1,3-PrD (12%) 7-fold 9 fold Ethylene glycol (12%) 10-fold 8-fold DMSO(6%) 12-fold 9 -fold Ethanol (12%) 25-fold 9-fold Isopropanol (12%)16-fold 10-fold Betaine (1.3M) 12-fold 13-fold Glycerol (12%) 4-fold5-fold

Table 2 shows enhancement of ligation by 6% 1,2-PrD and subsequenttransformation whether the insert being cloned is a synthetic cassettemade by annealing oligonucleotides or is a PCR amplicon. This test useda commercially available cloning vector from Qiagen, Valencia, Calif.(#231124) specifically designed to clone DNA fragments with single-base3′A overhangs.

TABLE 2 Cloning Enhancement Cloning Enhancement Insert (chemicaltransformation) (electroporation) 68mer cassette 14-fold 2-foldfeaturing No 5′ PO₄, 3′ A overhang 513 pb human At least 72-fold 11-foldgenomic amplicon (0 colonies vs. 72 using Taq colonies) DNA polymerase

The enhancement of ligation of double stranded or single strandedpolynucleotides has been shown here to be compatible with otherreactions which may be desirably conducted in a single tube. Forexample, site directed mutagenesis as described in Example 7 below,permits at least three different enzyme reactions (a restrictionendonuclease, a ligase and a PNK) to occur in a single reaction tube.

Small molecule ligase enhancers enable the development of a fast librarypreparation protocol with low nanogram polynucleotide input for multipleNext Generation Sequencing (NGS) platforms. Major improvements include:increased ligation efficiency of a ligase by one or more small moleculeenhancers; and tolerance of a ligase to dATP inhibition in the presenceof these small molecules. FIG. 6 shows the beneficial effects of smallmolecule ligation enhancers on T4 DNA ligase. Another application is theuse of long hairpin adaptors which are normally challenging to ligate topolynucleotide fragments using a ligase. These are readily ligated tothe polynucleotide fragments in the presence of small molecule enhancerswith increased ligation efficiency.

All references cited herein are incorporated by reference.

Examples Example 1 Ligation Efficiency Assay

A. Ligation Reactions

The reaction mixtures contained DNA at 50-100 ng and 1,2-PrD atdifferent concentrations. 1 μl of regular concentration (NEB, Ipswich,Mass., #M0202; 400 units) or 1 μl of high concentration T4 DNA ligase(NEB, Ipswich, Mass., #M0202; 2000 units) was then added to a finalvolume of 21 μl in 1× T4 DNA Ligase Buffer (NEB, Ipwich, Mass., #B0202)or 1× Quick Ligase™ Buffer (NEB, Ipswich, Mass., #B2200).

A standard assay uses 100 ng DNA, 1 μl of T4 DNA ligase, 21 μl 1× T4 DNAligase buffer and varying amounts of enhancer. However, we have alsotested and compared (a) various concentrations and types of DNA ligase,and (b) various ligation buffers (specifically, regular and QuickLigase) and found consistent enhancement of ligation at 1%-12% 1,2-PrD.

Ligation reactions were incubated for 10 min at room temperature (25°C.) unless otherwise stated, then placed on ice for 2 min. In someexperiments the ligase was heat-killed by incubation at 65° C. for 20min. The ligation reactions were analyzed by 1% agarose gelelectrophoresis to identify DNA species and determine the efficiency oftransformation.

B. Efficiency of Transformation Determinations

A volume of 2 μl from each ligation reaction was incubated with 50 μl ofchemically competent (NEB, Ipswich, Mass., #C2987I) or electrocompetent(NEB, Ipswich, Mass., #C2989K) DH5 alpha E. coli cells.

For chemically competent cell transformations, the cell-DNA mixture wasincubated on ice for 30 min, heat-shocked at 42° C. for 30 sec, thenreturned to ice for 5 min. A volume of 950 μl SOC medium (2% vegetablepeptone or Tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mMMgCl₂, 10 mM MgSO₄ and 20 mM glucose) was added to the cell-DNA mixture,followed by a one hr 37° C. outgrowth period with agitation. A volume of25-50 μl of appropriately diluted cells in SOC was spread onto Rich Agarplates (composition per liter: 10 g soy peptone, 5 g yeast extract, 10 gNaCl, 1 g MgCl₂-6H₂O, 1 g dextrose, 15 grams agar) supplemented with 100μg/ml ampicillin. Plates were inverted and incubated for 16 hrs at 37°C., followed by quantification of total colony numbers.

For electrocompetent cell transformation protocols, the cell-DNA mixturewas subjected to electroporation, immediately followed by the additionof 950 μl of pre-warmed 37° C. SOC medium, followed by a one hour 37° C.outgrowth period with agitation. Dilution, plating, incubation andcolony quantification were performed as described in Example 1A above.

The number of colonies per ligation condition correlated with theformation of circularized DNA constructs during the ligation reaction.

Example 2 Enhanced Ligation of DNA with a Single-Base Overhang

Linearization of the pUC19 plasmid was achieved using AhdI to generatesingle-base overhangs as described in FIG. 3A. Ligation of the cleavedDNA was performed in a 20 μl ligation reaction containing 100 ng of DNAin the presence and absence of 1,2-PrD and 400 U of T4 DNA ligase wasincubated for 20 min at 16° C., then heated to 65° C. for 45 min toinactivate the ligase. Transformation of cells with the ligated DNA wasperformed using chemically competent NEB Turbo™ cells (NEB, Ipswich,Mass., #C2984H) as described in Example 1 using 2 μl of the ligation mix(containing 10 ng DNA) with an outgrowth period of 20 min. The resultswere that 1,2-PrD increased transformation levels (see FIGS. 1A-C).

Example 3 Enhanced Ligation of DNA with Blunt Ends

The effectiveness of the ligation enhancer was also established forblunt-ended DNA as follows: 20 μl reactions were set up containingtwo-fold serial dilutions of 2000 units of T4 DNA ligase, 200 ngPhiX174-HaeIII digest (NEB, Ipswich, Mass., #N3026), and regular ligasebuffer with 200 ng phiX174-HaeIII blunt-ended fragments per 20 μl, inthe absence and presence of 6% 1,2-PrD. The reactions were incubated for30 min at room temperature (25° C.) followed by electrophoresis on a 1%agarose gel. 1,2-PrD enhanced T4 DNA ligase activity 4-fold in thistiter reaction.

Example 4 No Enhancement with 1,2-PrD Alone as Determined by MockLigation Reactions with Supercoiled DNA

Mock DNA ligations were set up with 1 ng of supercoiled pUC19 DNA in 1×regular DNA ligase buffer in the presence and absence of 2000 units T4DNA ligase and with or without 12% 1,2-PrD. Ligation reactions wereconducted using the standard protocol in Example 1 and transformationswith chemically competent DH5 alpha E. coli cells. 40 μl of a 1:20dilution of the 1 ml SOC outgrowth containing 2 pg supercoiled DNA wasplated onto agarose plates and incubated overnight. The number oftransformants was not statistically altered by the presence of 1,2-PrD,whether or not the T4 DNA Ligase was present.

Table 3 shows the results of a control experiment where 1,2-PrD does notaffect nor interfere with transformation using a supercoiled DNA form ofthe plasmid pUC19. This illustrates the enhancement is at the ligationstep and not the transformation step.

TABLE 3 T4 DNA Ligase 1,2-PrD Transformants from Levels Concentration 2pg SC DNA None  0% 134 None 12% 126 2000 U  0% 69, 92 (duplicatereactions) 2000 U 12% 82, 73 (duplicate reactions)

Example 5 Enhanced Ligation Efficiency of One, Two or Three DNAFragments into a Linearized Vector and Recircularization

Single fragment ligation (recircularization), two fragment ligation(cloning) and three fragment ligation (assembly) reactions wereperformed in the presence of 1,2-PrD (see FIGS. 3A-C). Restrictiondigests of pUC19 plasmid constructs were performed to completion bymonitoring the reactions using 1% agarose gel electrophoresis. Uponcompletion all restriction enzymes were heat-inactivated by incubatingthe reaction for 20 min at 65° C. The DNA was purified by use ofQiaQuick® (Qiagen, Valencia, Calif.) spin columns and quantified bycomparison to known DNA standards by 1% agarose gel electrophoresis. Thegel electrophoresis analysis shown in FIGS. 4A-C showed the productsmade with single (linearized), two and three fragment ligation reactionsusing restricted pUC19 plasmid DNA. Ligation reactions were performed asdescribed in FIGS. 3A-C using 100 ng of DNA that had been restricted byAhdI, BciVI or BccI to leave a variety of single-base overhangs. Fromeach 21 μl ligation reaction, 2 μl was used for transformation asanalyzed in Table 4 and 19 μl was run on a 1% agarose gel prepared withtris-borate as described in FIGS. 4A-C. These figures show that 1,2-PrDhad a significant beneficial effect on the amount of ligatedcircularized plasmid as indicated by arrow 5 for each plasmid testsystem. This is the highly transformable DNA circularized form.

Table 4 below shows the results of ligations performed on the constructsdescribed in FIGS. 3A-C as described in the basic protocols using 1× T4DNA Ligase Buffer and 100 ng of pUC19 DNA that had been restricted aslisted in column 1 (and diagrammed in FIGS. 3A-C), purified withQiaQuick® spin columns and quantified against known DNA standards(lambda-HindIII fragments). The ligations took place with and without12% 1,2-PrD present for 10 min at room temperature (25° C.) with 2000units T4 DNA ligase. A volume of 2 μl from each ligation reaction wastransformed into chemically competent DH5 alpha cells as described inExample 1. A volume of 40 μl of a 1:20 dilution of the outgrowth in SOCmedium was plated on medium Rich Agar plates supplemented withampicillin. This volume corresponded to 20 pg of DNA from the originalligation reaction.

TABLE 4 Resultant Resultant Ligation/ Nucleotide Colonies ColoniesSubsequent DNA Identity at (Transfor- (Transfor- Transfor- Fragments theSingle mation mation mation Used for Base Efficiency) − Efficiency) +Enhance- Ligations Overhang 1,2-PrD 1,2-PrD ment 1 fragment 3′-C, 3′-G84 (4.2 × 1027 (51.4 × 12-fold (AhdI- 10e⁶/μg 10e⁶/μg linearized DNA)DNA) pUC19) 2 fragments 3′-C, 3′-G 0 35 (1.8 × At least (BciVI-pUC193′-A, 3′-T 10e⁶/μg 35-fold DNA) 3 fragments 5′-C, 5′-G 2 (1.0 × 37 (1.85× 18-fold (BccI-pUC19) 5′-C, 5′-G 10e⁵/μg 10e⁶/μg 5′-A, 5′-T DNA) DNA)

Example 6 Comparison of the Numbers of Transformants Achieved in thePresence or Absence of 1,2-PrD in T4 DNA Ligase Buffer and Quick Ligase™Buffer Containing PEG 6000

Ligations were performed in both buffers using 50 ng of AhdI-linearizedpUC19 double-stranded DNA (containing single-base overhangs) followingthe conditions described in Example 1. A total of 10 pg DNA (40 μl of a1:20 dilution of the SOC outgrowth) was used per plate and transformantswere counted after 16 hrs at 37° C. FIG. 5 shows that even for the mostdifficult to ligate double-stranded DNA ends, 1,2-PrD in regular ligasebuffer achieved the level of transformation expected or greater than inPEG-containing (quick ligase) buffers.

Example 7 Ligation of Blunt-Ended DNA and DNA with T/A Overhangs Using aPlurality of Small Molecule Enhancers

Reaction conditions were utilized according to Example 1 for T4 ligasein Quick Ligase buffer, and for Taq ligase reaction buffer (NEB,Ipswich, Mass.). In addition to 5-10% PEG 6000 5%-15% glycerol and 1-8%1,2-PrD were included in the reaction mixtures together with blunt-endeddouble-stranded DNA or DNA with a T/A overhang. The ligated DNA wastransformed into host cells using chemically competent cells asdescribed in Example 1. An improvement of at least 25% in efficiency ofligation was observed in multiple samples in which glycerol and 1,2-PrDwere included in addition to PEG 6000 compared with ligationefficiencies in the presence of PEG 6000 only as a control. When theligated DNA was introduced into host cells using electroporation (seeExample 1), at least a 50-fold improvement was observed over thecontrol.

Example 8 A Method for Phosphorylating and Ligating Blunt-Ended Productsfrom a High-Fidelity Amplification Reaction Using a Multi-Enzyme Mixture

A mixture of enzymes comprising 0.01-200 units/μl DpnI, 4-200 units/μlT4 ligase, 0.01-20 units/μl T4 PNK, buffer, 3-10% 1,2-PrD, 3-10% PEG6000 and 2-20% glycerol in a ligase buffer as described above was addedto the PCR amplification product obtained using non-phosphorylatedpromoters, unmodified end to end primers and plasmids containing a DNAof interest.

In a single-reaction tube, the PCR product was phosphorylated using T4PNK and the ends were ligated to form a closed circle using T4 DNAligase or Taq ligase. The parental template was cleaved with DpnI toreduce the background of undesired colonies.

The circularized product was used to transform cells. A significantlyimproved number of colonies of transformed host cells were obtainedcompared to controls absent the small molecule ligation enhancer and theability to remove template, phosphorylate and ligate in a single stepsignificantly enhanced the efficiency of the entire site-specificmutagenesis reaction.

1. A composition comprising a ligase and one or more small moleculeligation enhancers having a molecular weight of less than 1000 daltons.2. A composition according to claim 1, further comprising a ligasereaction buffer in which the one or more small molecule enhancers arecontained at a concentration of 1%-50% v/v, the ligase reaction bufferbeing capable of enhancing, by at least 25%, intramolecular orintermolecular ligation of a polynucleotide fragment or fragments.
 3. Acomposition according to claim 1, wherein the one or more small moleculeligation enhancers are selected from an optionally substituted straightor branched chain diol or polyol containing 2 to 20 carbons, alcohols,zwitterionic compounds and polar aprotic molecules.
 4. A compositionaccording to claim 3, wherein the optionally substituted straight orbranched diol or polyol is selected from the group consisting of1,2-ethylene glycol, 1,2-propanediol (1,2-PrD), 1,3-propanediol(1,3-PrD), glycerol, pentaerythritol, sorbitol, diethylene glycol,dipropyleneglycol, neopentyl glycol, 2-methyl-1,3-propanediol,2,2-dimethyl-1,3-propanediol, 1,3-butanediol, 1,4-butanediol1,5-pentanediol, 1,6-hexanediol, 1,9-nonanediol, 1,12-dodecanediol,2,2′-dimethylpropylene glycol, 1,3-butylethylpropanediol,methylpropanediol, methylpentanediols, propylene glycol methyl ether,dipropylene glycol methyl ether, tripropylene glycol methyl ether,propylene glycol isobutyl ether, ethylene glycol methyl ether, ethyleneglycol ethyl ether, ethylene glycol butyl ether, diethylene glycolphenyl ether, and propylene glycol phenol ether.
 5. A compositionaccording to claim 3, wherein the alcohol is an optionally substitutedstraight or branched aliphatic primary, secondary, or tertiary alcoholchain alkylene group or polyvalent branched chain alkyl group,containing from 2 to 20 carbons.
 6. A composition according to claim 3,wherein the zwitterionic compound is optionally substituted, straight orbranched, containing from 2 to 50 carbons and further comprising anammonium, phosphonium, sulphonium cationic group, carboxylate, phosphateor a sulfate anionic group.
 7. A composition according to claim 3,wherein the polar aprotic molecule is selected from the group consistingof N-alkylcaprolactams, dimethylcaprylic/capric amides,N-alkylpyrrolidones, diphenyl sulfone, dimethyl sulfoxide (DMSO),N,N′-dimethyl imidazolidin-2-one, acetonitrile, acetone, diglyme,tetraglyme, tetrahydrofuran, dimethylacetamide, and dimethylformamide.8. A composition according to claim 1, wherein the one or more smallmolecule enhancers comprise a propylene glycol and an ethylene glycolether.
 9. A composition according to claim 1, wherein the one or moresmall molecule enhancers are selected from the group consisting of:1,2-ethylene glycol, 1,2-PrD, 1,3-PrD, isopropyl alcohol,1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol.
 10. A compositionaccording to claim 1, wherein the small molecule enhancer is selectedfrom the group consisting of: a quaternary ammonium or phosphoniumcation and a carboxylate anion.
 11. A composition according to claim 1,wherein the small molecule enhancer is selected from the groupconsisting of: 1,2-PrD, 1,3-PrD, ethylene glycol, ethanol, isopropanol,and betaine.
 12. A composition according to claim 1, wherein the smallmolecule enhancer is DMSO.
 13. A composition according to claim 1,wherein the small molecule enhancer is 1,2-PrD.
 14. A compositionaccording to claim 1, further comprising polyethylene glycol.
 15. Acomposition according to claim 1, wherein the amount of the smallmolecule enhancer is in the range of 1%-20% v/v.
 16. A compositionaccording to claim 1, further comprising at least two nucleic acidfragments wherein the nucleic acid fragments are double-stranded DNAhaving blunt ends for ligation to each other and the ligase is a DNAligase.
 17. A composition according to claim 1, further comprising atleast two nucleic acid fragments where the nucleic acid fragments aredouble-stranded DNA having single-base overhangs for ligation to eachother; and the ligase is a DNA ligase.
 18. A composition according toclaim 1, wherein the DNA ligase is selected from the group consisting ofT3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq Ligase, Ampligase, E.coli ligase and an Sso7-ligase fusion protein.
 19. A composition,according to claim 1, comprising: 0.01-200 units/μl restrictionendonuclease, 0.01-2000 units/μl ligase, 0.01-200 units/μlpolynucleotide kinase, in a buffer optionally containing PEG5000-10,000.
 20. A composition according to claim 19, wherein the one ormore small molecule ligation enhancers are 1,2-PrD and glycerol.
 21. Areaction mixture, comprising a composition according to any of claims1-18, and a ligation buffer in the absence of polyethylene glycol (PEG).22. A method of enhancing ligation between nucleic acid fragments, themethod comprising: (a) mixing a composition according to claim 1 with aplurality of nucleic acid fragments in a ligation buffer; and (b)permitting ligation wherein the efficiency of ligation is enhanced by atleast 25% compared to the efficiency of a ligation in the absence of thesmall molecule enhancer.
 23. A method according to claim 22, whereinligation is intramolecular.
 24. A method according to claim 22, whereinligation is intermolecular.
 25. A method according to claim 22, whereinthe plurality of nucleic acid fragments are double-stranded DNA withblunt ends, single-base overhangs or staggered ends.
 26. A methodaccording to claim 22, wherein the efficiency of ligation is enhanced byat least 4-fold as determined by electroporation of host cell.
 27. Amethod for creating a polynucleotide library, comprising: adding acomposition according to claim 19 or 20 to a polynucleotideamplification product; and allowing intramolecular ligation to occur fortransforming competent host cells.
 28. A method according to claim 27,wherein transforming competent cells further comprises electroporation.